The present invention relates to the field of fungal biotechnology, more particularly to strong gene expression systems in species in the Pucciniomycotina and Ustilaginomycotina subphyla.
The publications and other materials used herein to illuminate the background of the invention, and in particular, cases to provide additional details respecting the practice, are incorporated by reference, and for convenience are referenced in the following text by author and date and are listed alphabetically by author in the appended bibliography.
The Pucciniomycotina is a subphylum of fungi in the phylum of Basidiomycota (Kirk et al., 2008). It holds many species that have important industrial applications. For example, a number of species in the Rhodosporidium and Sporidiobolus genera, such as Rhodosporidium toruloides (also known as Rhodotorula gracilis, Rhodosporidium glutinis, Rhodotorula glutinis, Torula koishikawensis and Torula rubescens) and Sporobolomyces salmonicolor, are oil-rich single-cell yeasts capable of high density fermentation (Hu et al., 2009; Meng et al., 2009). These species hold great potential as a host for the production of long chain hydrocarbons, such as triacylglycerol (TAG, or fat), fatty acid esters (biodiesel), fatty alcohols, alcohols, lactones, terpenoids and vitamins (Wu et al., 2010a; Wu et al., 2010b; Zhao et al., 2010a; Zhao et al., 2010b). In another example, species in Ustilaginomycotina subphylum, in particular, Ustilago and Pseudozyma genera, are known to produce glycolipids, which may function as a surfactant or fungicide (Hewald et al., 2005; Teichmann et al., 2010).
Promoters that are able to drive strong gene expression, either constitutively or inducibly, are critical for the development of biotechnological applications of a microorganism. WO 2012/169969, incorporated by reference herein in its entirety, describes several polynucleotide sequences derived from the upstream region of glyceraldehyde phosphate dehydrogenase gene (GPD1), translation initiation factor gene (TEF1), and putative stearoyl-CoA-delta 9-desaturase gene (FAD1) of selected fungal species that are able to function as a strong promoter of gene expression in Pucciniomycotina and Ustilaginomycotina subphyla. As repeated use of the identical or highly homologous promoter risks genome instability, epigenetic and genetic modification of chromatin resulted from repeat induced point mutation (RIP) or RNA silencing (Horns et al, 2012), an enlarged promoter pool is highly desirable for Pucciniomycotina and Ustilaginomycotina subphyla, wherein functionally verified promoters are scarce.
Promoters are DNA sequences located in the 5′ region adjacent to the transcriptional start site. It houses a combination of cis-acting DNA elements that act to interact with transcription factors by activating or repressing transcription of RNA polymerase. To date, genome shotgun sequences have been published for Rhodotorula glutinis ATCC 204091 (GenBank Accession: GL989638.1), Rhodosporidium toruloides MTCC 457 (GenBank Accession: PRJNA112573), Rhodosporidium toruloides NP11 (GenBank: ALAU00000000.1) and draft genome sequences have been published for Rhodotorula graminis WP1 (http://genome.jgi-psf.org/Rhoba1_1/Rhoba1_1.home.html) and Sporobolomyces roseus (http://genome.jgi-psf.org/Sporo1/Sporo1/Sporo1.home.html). RNA-Seq, proteomic and genome shotgun data released for Rhodosporidium toruloides NP11 (Zhu, Z., et al, 2012) are not able to define the sequence of functional promoters because the activity of a promoter is influenced by several factors, such as the location of 5′ and 3′ ends, posttranscriptional silencing, influence of intron, etc. The activity of a promoter in a heterologous host species is even more unpredictable.